The success of the genome projects has resulted in the identification of a vast number of open reading frames (ORFs), which potentially code for proteins. The main problem is now to assign functions to the forty to sixty percent of the ORFs in a genome for which no function can be allocated. Two analytical chemical approaches to this can be identified: transcriptomics and proteomics. Transcriptomics analyses the expression levels of the various mRNA species being transcribed using either a ‘gene-chip’ or a SAGE approach. This is useful for identifying under which conditions a particular protein is being expressed but provides little direct information as to function. Proteomics, the direct quantitation and analysis of expressed proteins, provides a more direct approach to function definition. Proteomics can be divided into two areas: Expression Proteomics attempts to define all the proteins being expressed in a cell and their post-translational modifications and how these change under various conditions. Cell-Map Proteomics attempts to define the subcellular location of a protein and with which other proteins it is interacting. It is this field which the inventors wish to address in this grant application.
Traditionally, protein-protein interactions have been analysed by the isolation of protein complexes by ‘soft’ non-denaturing physico-chemical methods such as centrifugation or affinity based isolations. This approach has been facilitated by the use of mass spectrometry to analyse the purified protein complexes either as mixtures or after separation by SDS-PAGE. The method suffers from several drawbacks, the main one being the stability of the complex under the conditions of purification and a lack of a general purification approach to allow a systematic analysis of many proteins. The latter problem has been successfully addressed by the development of the TAP procedure by the group of Bernard Seraphin at EMBL (Rigaut et al., Nat Biotechnol 1999 October; 17(10): 1030–2 and Puig et al., Methods 2001 July; 24(3): 218–29). ORFs are modified so that the proteins they encode contain two affinity purification (TAP) tags, which allow the labelled protein to be rapidly purified to homogeneity. The resulting complex is then analysed by mass spectrometry to identify the co-purifying proteins.
An alternative approach to protein-protein interaction analysis has been the development of the yeast and bacterial two- and three-hybrid systems (Fromont-Racine et al., Nat Genet 1997 July; 16(3): 277–82 and Uetz et al., Nature 10 Feb. 2000; 403(6770): 623–7). This has allowed genome-wide scans of all protein-protein interactions in a genome to be carried out. The main drawback here is that transient interactions and those induced by ligands or phosphorylation are not amenable to analysis by this method. The TAP purification and two-hybrid system methods do not allow one to define which protein is interacting with which other protein in the complex and which parts of the two proteins are involved in this interaction.
EP 0 778 280 (Isis Innovation Limited) relates to a reagent for use in biological and chemical analyses. More specifically, the reagent is comprised of at least two analyte groups linked to a tag comprising one or more reporter groups adapted for detection by mass spectrometry (MS). More specifically, the group for MS detection is a tertiary amino group, which increases sensitivity and which does not allow generation of a specific ion for parent ion scanning. Hence, the disclosed reagent cannot be used in parent ion scattering.
Further, WO 00/02893 (Brax Group Limited) relates to a method of characterising an analyte, which method comprises to provide a compound in which the analyte is attached by a cleavable linker to a mass marker relatable to the analyte; to cleave the mass marker from the analyte; and to identify the mass marker. More specifically, the marker is a metal ion-binding moiety. To achieve a high ionisation, the labels disclosed are pre-ionised.
Accordingly, there is a need for alternative methods in the field of proteomics, solving the posed problems and providing new opportunities to gain more detailed information about molecule-molecule interactions.